Epigenetic changes can influence gene expression through various mechanisms including DNA methylation and chromatin modifications. In particular, histones can be subject to many alterations associated with an increase or a decrease in gene transcription. Epigenetic regulation modifies individual genes but it can also affect a whole chromosome, for example in the case of X chromosome inactivation. The fundamental genetic difference between the sexes (male, XY;female, XX) has led to the necessity of dosage compensation mechanisms: up-regulation of the active X chromosome in both sexes and inactivation of an X chromosome in females. Although most genes on the inactive X (Xi) are silenced, some have attained mechanisms that lead to their escape and subsequent expression from the Xi. The importance of genes that escape X inactivation is illustrated by the phenotypic defects found in patients with Turner syndrome associated with a single X chromosome. X inactivation is associated with repressive histone marks including methylation of lysine 27 at histone H3. We proposethat removal of this repressive histone modification by the histone de-methylases UTX and JMJD3 is an essential part of escape from X inactivation. We will pursue the following three aims: Aim1. To determine whether histone H3K27 demethylases are involved in initiation of escape from X inactivation during early development through demethylation of H3K27;Aim2. To determine whether histone H3K27 demethylase depletion alters the onset and maintenance of escape from X inactivation;Aim3. To identify proteins bound to or associated with UTX during differentiation that may aide in escape from X inactivation. Our studies will help identify the role of histone demethylases in epigenetic regulation of the X chromosome and of the genome in general. Our studies are relevant to the understanding of epigenetic dysregulation in diseases such as cancer and aging.